1. Field of the Invention
The present invention relates to a process for cutting sections from a probe for microscopic analysis by using an ultramicrotome device.
2. Description of Related Art
Microtomes and ultramicrotomes are used to cut thin respective ultra-thin sections from a sample for microscopic analyses. The sample is mounted on a cross-slide which can be advanced horizontally in steps according to the desired thickness of the sections and vertically for performing the cutting operation. A cutting blade with a horizontal cutting edge is mounted on a holder. Microtomy is concerned with a range of thickness of 0.5 to 50 μm of the sections and is mainly used for optical microscopy. Ultramicrotomy is concerned with a range of thickness of 10 to 100 nm of the sections. This range of thickness is required for transmission electron microscopy. Ultramicrotomy has proved to be a very fast and efficient technique not only for TEM but also for surfacing samples for STM and AFM.
In microtomy mainly steel blades are used for cutting. German Patent No. 913 112 discloses an older type of a microtome in which the cutting blade is horizontal and the sample advances upwardly in steps between the cuts. The blade is fastened between two parallel leaf springs and driven by magnets for oscillating movement parallel to the cutting edge. The cutting edge of a steel blade is relatively rough when viewed under an electron microscope and relatively blunt. With the oscillating motion of the blade, therefore, a sawing action is achieved: the jags of the cutting edge act like saw teeth.
This sawing action of the blade in a microtome is also described in the DDR Patent No. 156 199, in which the blade is driven by an electroacoustical transducer at high frequency, and in the Belgian Patent No. 440 928 which uses an ultrasound emitter to oscillate the blade.
In ultramicrotomy the sections are so thin that extreme care must be taken to shield the ultramicrotome from all possible external and internal vibrations because they would adversely affect the cutting result. It therefore seemed impossible to transfer the sawing action of the cutting blade known from microtomy to an ultramicrotome. For this reason much sharper and perfectly rectilinear cutting edges are required in ultramicrotomy. This has been achieved by cutting blades of diamond. U.S. Pat. No. 4,697,489 describes a holder with such a diamond cutting blade for ultramicrotomes. With the perfectly rectilinear cutting edge, even when viewed under an electron microscope, of a diamond blade no sawing action can be achieved as with steel blades.
For the ultramicrotomy at room temperature, usually the cutting is performed on a knife mounted in a boat which contains water. The water forms a horizontal surface behind the cutting edge of the knife. Due to the surface tension the sections float on the water surface and can be collected. The water acts as a lubricant during sectioning process.
However, in ultramicrotomy a different problem arises which does not occur in microtomy: the problem of section compression. This phenomenon occurs at a thickness of the sections below 100 nm. Depending on the mechanical properties of the sample (flexibility) and on the sectioning angle φ of the knife the sections undergo considerable distortion (compression) during cutting (FIG. 8). In FIG. 8, 1 designates the diamond blade or knife with the cutting edge 2. 3 is the sample. The sample 3 may be one of a great variety of industrial or biological samples. A is the vertical movement of the sample 3. 4 is the cut section floating on a waterbed 5. 6 designates the direction of compression in the section 4. 7 is a region of intense shearing, and 8 is the region of compression in the sample 3.
Water sensitive samples 3 have to be cut dry. Due to the missing lubrication and to the friction on the knife surface the sections 4 are even more compressed as the ones cut on water. In cryo-UM most samples have to be cut dry. The amount of compression depends on different factors:                The sectioning angle of the knife.        The hardness of the sample.        The triboelectrical properties of the sample.The most critical factor is the sectioning angle φ. The sectioning angle φ is the sum of the wedge angle β of the knife 1 and the clearance angle δ. It was shown that reducing the wedge angle β results in a reduction of compression. However, the wedge angle β may not be reduced ad infinitum. We have found an angle of 30° to be a limit. A further reduction of the wedge angle results in a lower cutting edge 2 quality and in a considerably shorter service time of the knife 1. In cryo-UM the compression in sections was found almost equal with the sectioning angle φ. Therefore, a knife 1 working with a sectioning angle φ of 40° (wedge angle β 30°, clearance angle δ 10°) would result in a compression in the sections 4 of approximately 40%.        